FARMACIA, 2017, Vol.

65, 4
ORIGINAL ARTICLE

THE INFLUENCE OF A NEW RUTIN DERIVATIVE IN AN

EXPERIMENTAL MODEL OF INDUCED
HYPERHOMOCYSTEINEMIA IN RATS

CRISTIANA FILIP1, ELENA ALBU2*, DAN LUPACU3, NINA FILIP1

Grigore T. Popa University of Medicine and Pharmacy, Iai, Romania

1
Department of Biochemistry,
2
Department of Pharmacology,
3
Department of Pharmaceutical Chemistry

*corresponding author: elenaalbu@yahoo.com

Manuscript received: February 2017

Abstract
Cardiovascular diseases are a major cause of deaths today. It is generally accepted the involvement of three major risk
factors: hyperlipidaemia, hyperhomocysteinemia and the molecular reactive species excess. Therapeutic approaches try to
reduce the levels of lipids and homocysteine and to keep the balance between the oxidation/reduction system. We studied a
new rutin derivative coded L103 that may determine the lowering of the lipid level and antioxidant properties. L103 contains
a pyridine group attached to a rutin molecule. We studied the influence of L103 on the plasmatic levels of cholesterol, total
antioxidant status and homocysteine in hyperhomocysteinemia experimentally induced in a rat model through methionine
loading. The obtained data suggest that L103 succeeds to prevent the decline of the total antioxidant status, keeps cholesterol
within normal ranges but has minor effects on the homocysteine levels. Despite the low influence on homocysteine, L103
confers protection against two of the major risk factors incriminated in cardiovascular diseases.

Rezumat
Bolile cardiovasculare sunt o cauz major a deceselor n prezent. Este general acceptat implicarea a trei factori de risc
majori n bolile cardiovasculare: hiperlipidemia, hiperhomocisteinemia i speciile reactive la nivel molecular. n terapie se
ncearc scderea nivelurilor lipidice i homocisteinei precum i meninerea statusului antioxidant. Am studiat un nou derivat
de rutin codificat L103, cu posibile proprieti antioxidante i hipolipidice. L103 conine o grupare piridinic ataat la o
molecul de rutin. Am studiat influena L103 asupra nivelurilor plasmatice ale colesterolului, homocisteinei i a statusului
antioxidant total n hiperhomocisteinemia indus experimental la obolan prin ncrcarea cu metionin. Datele obinute
sugereaz c L103 previne declinul statusului antioxidant, menine colesterolul n limite normale, dar are efect sczut asupra
nivelurilor de homocistein. n ciuda influenei slabe asupra homocisteinei, L103 confer protecie mpotriva a doi dintre
factorii de risc majori din bolile cardiovasculare.

Keywords: severe/intermediary hyperhomocysteinemia, total antioxidant status, cholesterol, rutin, rat model

Introduction preserving the redox balance. Our study investigates

the influence of a new rutin derivative coded L103,
Atherosclerosis is considered a "hotbed" epidemic
on the previous mentioned cardiovascular risk factors,
in developed countries, if we consider the increased
in a rat model with experimentally induced HHcy
number of people affected [3]. Proven major cardio-
through methionine loading. L103 contains a pyridine
vascular risk factors are hyperlipidaemia, hyperhomo-
group (similar to niacin) chemically bound to a rutin
cysteinemia (HHcy) and excess reactive species.
molecule. Niacin exerts antihypertensive effects at
Elevated serum lipids initiate a series of reactions
low doses, hypolipidemic effects at high doses [13]
that finally generates excessive reactive species.
and is used in the cardiovascular therapy [16]. Rutin
Oxidised LDL fractions trigger the onset of the
is a citrus flavonoid glycoside presenting antioxidant
endothelial damage [15]. Elevated homocysteine
activity and inhibiting platelet aggregation [4]. Based
(Hcy) levels, even slightly above the upper limit [8, 17]
on the pharmacology of the initial molecules, niacin
affect the endothelial function, cause vasoconstriction
respectively rutin, we estimated for L103 hypolipidemic
and initiate the coagulation cascade by generating
and antioxidant activities. The influence of L103 on
reactive species after the autooxidation of homocysteine
the hyperhomocysteinemia-induced rats was determined
[7]. Thus, hyperhomocysteinemia is an aggravating
by measuring plasma concentration of cholesterol,
factor for hyperlipidaemia. Therapy should be targeted
homocysteine and by the evaluation of the total
to both lowering homocysteine and lipid levels and
antioxidant status (TAS). The obtained data suggest
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that L103 prevents the increase of cholesterol, preserves Statistical analysis
the total antioxidant status, but has negligible effect The statistical analysis used the analysis of variance
on hyperhomocysteinemia levels. (ANOVA-one-way) and Turkey-Kramer multiple
comparisons; a coefficient p < 0.05 was considered
Materials and Methods to indicate a statistically significant difference within
or between groups.
Synthesis and analysis of the new rutin derivative
The new compound (L103) was synthesized from
Results and Discussion
rutin and 2-amino-pyridine, using a previously
described method [14]. The melting point was Chemistry
measured using an Electrothermal Mel-Temp apparatus The chemical name for L103 is 3-[[6-O-(6-deoxy-
and it is uncorrected. The IR spectrum was recorded -L-manopyranosyl)--D-glucopyranosyl]-oxy]-2-(3,4-
on a FT/IR Jasco 670 Plus spectrometer. The 1H- dihydroxy-phenyl)-5-hydroxy-7-(oxy-(-hydroxy-
NMR spectrum was recorded on a Bruker AC-300F, propyl-(amino-pyridin-2-yl))-4H 1-benzopyran-4-one.
300 MHz instrument using DMSO-d6 as solvent. The chemical structure of L103 consisting in a pyridine
The elemental analysis was performed on an Exeter group attached to rutin molecule is presented in
Analytical CE-440 elemental analyser. Figure 1.
Animals
The pharmacological study was performed on three OH
OH
groups of 10 adult Wistar male rats, weighing 150 -
N
200 g. The rats received standard food (containing NH CH2 CH CH2 O O
folic acid and vitamin B12) and water ad libitum. OH
All procedures were performed according to the O rham glu
European legislation concerning the care and use of OH O
animals for scientific purposes (Directive 86/609/ Figure 1.
EEC). Chemical structure of the rutin derivative coded L103
Experimental
Hyperhomocysteinemia was experimentally induced L103 characterization: Yellow crystalline powder
to all groups by oral administration of methionine (yield 70.32%), m.p. = 215 - 217C, IR (KBr, cm-1):
1.8 g/kg body weight (b.w.) single dose daily, for 3310 (linked OH), 2993 (N-H), 2922 (C-H), 1657
30 days [10]. Group I served as control and received (C=O aromatic ring), 1610 (aromatic structure), 1505
only methionine 1.8 g/kg b.w., for 30 days; Group (aromatic C=C), 1362, 1312, 1215, 1060 (C-O-C),
II received methionine similar to Group I and niacin 979 (pyridine ring), 812 (aromatic substitutes); 1H-
orally 50 mg/kg b.w. single dose daily, for 30 days; NMR (DMSO-d6) ppm: 8.10 (s, 1H, pyridine ring),
Group III received methionine similar to Group I 8.00 (s, 1H, pyridine ring), 7.92 (s, 1H, pyridine ring),
and orally L103 in 36.76 mg/kg b.w. single dose 7.82 (s, 1H, pyridine ring), 7.44 (s, 1H, aromatic),
daily, for 30 days. The dose for L103 represents 6.70 (d, 1H, aromatic), 6.30 (s, 1H, aromatic), 6.14
1/20 from 50% lethal dose (LD50) in rats. The blood (s, 1H, aromatic), 5.40 (s, 1H, H-1 glucosyl), 4.56 (s,
samples were taken from the retro-orbital plexus. 1H, H-1 ramnosyl), 3.88 (s, 2H, CH2N), 1.21 (s, 1H,
TAS, homocysteine and cholesterol concentrations NH); Elemental analysis: calculated for C35H40N2O17:
were determined at the beginning and at the end of C: 55.26; H: 5.29; N: 3.68; Found: C: 55.19; H: 5.35;
the experiment thus each group had its own control N: 3.72.
in the initial values. TAS and cholesterol concentrations Pharmacological study
were measured using a Randox kit. Total plasma Homocysteine concentrations determined in rat
homocysteine was determined by a HPLC validated plasma are presented in Table I.
method [2].
Table I
Homocysteine concentrations determined in rat plasma
Hcy (mol/L); Mean SD
N = 10 animals/group Initial Final
Group I - only methionine 9.692 0.692 31.056 0.616*
Group II - methionine plus niacin 10.555 0.668 28.06 0.540*
Group III - methionine plus L103 10.167 0.852 26.828 0.505*
* Statistical difference within group; Statistical difference between groups

The obtained data showed significant increase in moment compared to the initial moment, which
homocysteine level for all groups at the final confirm HHcy status.

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Data are consistent with the literature [11] and our and the control group in the intermediary one. Thus
previous studies [5]. Hcy levels in groups II and III for the situation of moderate /intermediary HHcy,
were significantly lower as comparing to the control as in our case, L103 fails to prevent the increase of
group at the end of the experiment (< 0.001 for both homo-cysteine levels. Even so, lowering Hcy level
groups), but Hcy is still over the upper limit (up to from intermediary to moderate hyperhomocysteinemia,
0.7 mol/L [1]). Between Group II and Group III might be a benefit for L103 administration.
there is no statistical difference in Hcy levels in the The total antioxidant status determined in rat plasma
end. Hcy concentrations in blood are classified as is presented in Table II.
follows: normal range 5 - 15 M; moderate 16 - 30 TAS concentrations were significantly decreased at the
M; intermediary 31 - 100 M; severe above 100 end as compared to the beginning of the experiment,
M [9]. According to this classification, groups II within all groups (p < 0.0001, p = 0.0064 respectively
and III belong to moderate hyperhomocysteinemia, p < 0.0001).
Table II
TAS status determined in rat plasma
TAS (mmol/L plasma); Mean SD
N = 10 animals/group Initial Final
Group I - only methionine 1.810 0.111 1.064 0.095*
Group II - methionine plus nicotinic acid 1.737 0.134 1.237 0.203*
Group III - methionine plus L103 1.859 0.049 1.469 0.116*
* Statistical difference within group; Statistical difference between groups

Regarding TAS status, between groups II and control radicals generated through the autooxidation of Hcy
there are no statistical differences at the end. The high concentrations [6, 12]. Group III presents
fact that TAS levels are slightly higher in Group II significantly higher TAS concentration as compared
versus control suggests a minor antioxidant effect to the control group at the end of the experiment
attributed to niacin. Literature shows that the oxidant/ (p = 0.0011). In fact, the drop in TAS concentrations
antioxidant activity of niacin depends on the time was the smallest among all three groups, suggesting
of exposure as follows: the antioxidant activities of a protective antioxidant activity attributable most likely
niacin in the later phases (3 weeks) of lipid per- to the rutin moiety. The comparison between groups
oxidation are much stronger than those in the II and III showed no statistical difference at the
earlier phases (1 week) [10]. Even if niacin has final moment.
been administered for 4 weeks, we assume that its Cholesterol concentrations determined in rat plasma
antioxidant capacity was exceeded by the free are presented in Table III.
Table III
Cholesterol concentrations determined in rat
Cholesterol (mg/dL); Mean SD
N = 10 animals/group Initial Final
Group I - only methionine 85.94 3.31 97.43 4.16*
Group II - methionine plus nicotinic acid 89.27 5.11 88.07 3.07
Group III - methionine plus L103 86.47 9.79 87.30 2.72
* Statistical difference within group; Statistical difference between groups

Cholesterol concentrations were significantly increased respectively p = 0.0008) suggesting the lipid-lowering
at the end of the experiment as compared to the activity for niacin as well as for L103.
initial one in the control group (p = 0.0013).
Literature shows [18, 19] that after methionine Conclusions
administration, cholesterol level increases because
The new rutoside derivative L103 presents anti-
of the stimulation of its hepatic synthesis, our data
oxidant properties and lowers the cholesterol levels
being consistent to that. Within groups II and III no
in the experimental induced hyperhomocysteinemia
statistic difference in cholesterol concentrations was
in rats. We assume that in the case of the moderate/
found when comparing initial to final moment. For
intermediary hyperhomocysteinemia, the decrease
Group II, this result is justified because niacin is a
of two aggravating risk factors (cholesterol and
well-known hypolipemiant drug. The similar behaviour
reactive species) justifies the use of L103. Since
for Group III suggests a nicotinic acid-like activity
L103 exerts similar lipid-lowering effect as niacin,
for L103. Both groups II and III exhibit significant
its administration might avoid the niacin side effects
decreased cholesterol levels as compared to the control
being a benefit in dyslipidaemia. The fact that L103
group at the end of the experiment (p = 0.0035

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pulls back homocysteine levels, even in a very small
amount, may be useful in cardiovascular disease.
Acknowledgement
This study was supported from scientific grant from
Romanian Education Ministry; program PN-II IDEI,
cod 1225, Grant no. 223/2007.
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